1. Field of the Invention
This invention relates generally to compositions and methods of regulating adipogenesis in cells. Specifically, the invention is directed to the utilization of nuclear receptors and their cofactors including PPARγ. Also related are polypeptides that enhance or inhibit PPARγ activation and the subsequently the transcription of genes responsible for adipogenesis. Also related are nucleotide sequences which express these polypeptides.
2. Description of the Related Art
Obesity, defined as a state of pathologically excessive adipose tissue mass, has been identified as an epidemic in the U.S. for more than two decades. Yet the numbers of overweight and obese adults and children continues to rise. Currently, the rates of both overweight and obesity in the U.S. are 61 percent and 14 percent in adults and children, respectively. Among U.S. adults aged 20-74 years, the prevalence of overweight (defined as BMI 25.0-29.9) has increased from 33 percent in 1980 to 35 percent of the population in 1999. The disease is associated with several serious health conditions including type 2 diabetes mellitus, heart disease, high blood pressure and stroke. It is also linked to higher rates of certain types of cancer. Obesity is an independent risk factor for heart disease, hypoxia, sleep apnea, hernia, and arthritis. Obesity is the seventh leading cause of death in the U.S. The total cost of obesity and related conditions by some estimates is $100 billion annually.
The development of obesity requires the continuous differentiation of new adipocytes throughout life, and this process of adipocyte differentiation from preadipocytes is called adipogenesis. Many studies have demonstrated the importance of transcription factors, especially nuclear receptors (NRs) and their coactivators, in adipocyte proliferation, gene expression regulation, and differentiation.
Nuclear receptors are a superfamily of transcription factors (TFs) that regulate the expression of target genes in response to steroid hormones and other ligands. To date, there are 48 NRs reported and each plays distinct or interrelated functions (Ruan et al. (2005) Kidney International 68, 2444-2461). However, recent studies have demonstrated that NR functions are modulated by a large group of proteins called coregulators. These coregulators include coactivators which promote transcription and corepressors which attenuate promoter activity when recruited into the promoter regions of specific genes (McKenna and O'Malley (2002) Endocrinology 143, 2461-2465; Glass (2006) J. Clin. Invest. 116, 556-560; Aranda and Pascual (2001) Physiol. Rev. 81, 1269-1304; McKenna and O'Malley (2002) Cell 108, 465-474; Smith and O'Malley (2004) Endocr Rev 25, 45-71; Darimont et al. (1998) Genes Dev. 12, 3343-3356; Xu (2005) Biochem Cell Biol 83, 418-428; O'Malley (2003) Nucl Recept Signal 1, e010; Lee et al. (2001) Cell Mol Life Sci 58, 289-297; Robyr et al. (2000) Molecular Endocrinology 14, 329-347).
Regulation of adipogenesis is central to conditions of overweight and underweight. Adipogenesis is a multistage process that includes expression of a complex set of transcription factors that initiate transcription of preadipocyte- or adipocyte-specific genes. This transcriptional cascade includes expression of key adipogenesis regulators, such as PPARs, CCAAT/enhancer binding protein (C/EBP), and the basic helix-loop-helix (bHLH) family of transcription factors such as ADD1/SREBP1c (Rosen et al. (2000) Genes Dev. 14, 1293-1307). These transcription factors activate genes encoding enzymes that are involved in lipid storage and transport such as adipocyte-specific fatty acid binding protein (aP2) and perilipin, and genes encoding secreted proteins or adipokines that modulate preadipocyte and adipocyte functions such as adiponectin and leptin. In adipocytes, PPARγ regulates the expression of genes involved in lipid synthesis, storage, and transportation (Berger and Moller (2002) Annu Rev Med 53, 409-435). PPARγ is essential to activate the promoters of acyl-CoA oxidase and aP2 and many other fat-cell specific genes (Kliewer et al. (1992) Nature 355, 446-449; Tontonoz et al. (1994) Cell 79, 1147-1156).
Coactivators play critical roles in this nuclear receptor directed adipogenesis program. The importance of coactivators in metabolism and adipogenesis are well documented (Castillo et al. (1999) EMBO J 18, 3676-3687; Puigserver et al. (1998) Cell 92, 829-839; Puigserver and Spiegelman (2003) Endocrine Rev 24, 78-90; Qi et al. (2003) J. Biol. Chem. 278, 25281-25284; Ge et al. (2002) Nature 417, 563-567; Andersen et al. (2005) J Med Genet 42, 402-407; Wang et al. (2006) Cell Metabolism 3, 111-122). Most coactivators identified to date interact with PPARγ through its C-terminal AF-2 domain or LBD domain, which is dependent on or enhanced by ligand binding, and mediated by the signature cofactor motif, LXXLL, (McInerney et al. (1998) Genes Dev. 12, 3357-3368). For example, coactivators SRC-1/NCoA-1, CBP/p300, pCAF, and TRAP220 interact with the LBD domain of PPARγ (Robyr et al. (2000) Molecular Endocrinology 14, 329-347; Castillo et al. (1999) EMBO J 18, 3676-3687; Nolte et al. (1998) Nature 395, 137-143; Zhu et al. (2000) J. Biol. Chem. 275, 13510-13516). Other coactivators such as PGC-1α interact with PPARγ through the PPARγ DNA-binding and hinge domains (Puigserver et al. (1998) Cell 92, 829-839). PGC-2 binds to the PPARγNB domain, which is independent of ligand and LXXLL motif binding. Many NR coactivators such as members of p160/SRC and PGC-1 family are highly versatile. They are expressed in a variety of tissues and coactivate a wide spectrum of NRs including PPARγ (McKenna & O'Malley (2002) Cell 108, 465-474; Puigserver et al. (1998) Cell 92, 829-839; and Lin et al. (2005) Cell Metab 1, 361-370). For example, PGC-1α is also a coactivator for estrogen receptor (ER)α. Multiple sites in PGC-1α govern its interaction with ERα, but the presence of an LXXLL motif is required for PGC-1α's ligand-dependent binding to ERα (Tcherepanova et al. (2000) J. Biol. Chem. 275, 16302-16308). Therefore, whether a ligand is required for the NR-coactivator interaction mainly depends upon whether LBD domain of NR is involved in the interaction.
Modulation of nuclear receptors, their cofactors, and their pathways will offer new avenues for therapeutic strategies to combat obesity and diseases associated with an overweight as well as underweight conditions. Therefore, the inventor has sought to address the issue of adipogenesis, through the use of the novel nuclear receptor coactivator and its ability to promote or inhibit transcription.